Methods and compositions for predicting chronic lung allograft dysfunction

ABSTRACT

Some embodiments are directed to a prognostic method for determining whether a subject is at risk of having the CLAD, comprising: measuring the expression level of POU2AF1 or BLK in a biological sample obtained from the subject; comparing the expression level of POU2AF1 or BLK with a predetermined reference value and concluding that the subject is at risk of having CLAD when the expression level of POU2AF1 or BLK is lower than the predetermined reference value.

FIELD OF THE INVENTION

The invention is in the field of lung transplantation, particularly, theinvention allows to identify whether a subject is at risk of developingbronchiolitis obliterans syndrome.

BACKGROUND OF THE INVENTION

Chronic lung allograft dysfunction (CLAD) is the main limitation oflong-term survival after lung transplantation. CLAD manifest mainly byan abnormal remodeling of the small airways resulting in progressiveairflow obstruction called Bronchiolitis Obliterans Syndrome (BOS)(1-3). A restrictive ventilatory process referred as RestrictiveAllograft Syndrome (RAS) has been described recently as another form ofCLAD (4). The prevalence of CLAD reaches 50% at 5 years (35% BOS and 15%RAS) of lung transplant recipients. Its late diagnosis, based upon thedecline of lung function, reveals an advanced degradation of theallograft. Prognosis is poor, with respectively 4 and 2 years mediansurvival for BOS and RAS after onset. Identification of harbingers ofCLAD in lung transplant recipients is thus necessary to allow proactiveand targeted strategies to harness the progression of the disease,before irreversible degradation of the allograft.

It is hypothesized that CLAD arises from repeated injuries from bothalloimmune and non-alloimmune mechanisms, generating fibrosis and airwayobstruction (5). Tracking these inflammation and fibrotic processes haslong been used to identify early signs of the disease. BAL neutrophilia,levels of regulatory T cells, chemokines/cytokines or matrixmetalloproteases (MMP) have thus been suggested as early biomarkers ofCLAD (6-10). More recently, expression profiling of lung biopsiespinpointed fibrosis-associated genes for the diagnosis or the predictionof CLAD (11). Yet, these invasive lung-centered approaches remainedhampered by the accessibility to biological samples and are thereforelimited for a routine monitoring of LTR. In blood, circulatingfibrocytes or cytokine concentration have been proposed as potentialbiomarkers (12-15). However, these studies concerned a limited number ofpatients and confirmation in follow-up studies are still missing.Consequently, none of these attempts have demonstrated yet enoughfeasibility and robustness to achieve clinical acceptance. Accordingly,there is a need to identify new methods that allows to explore CLAD andprovide early biomarkers of CLAD.

SUMMARY OF THE INVENTION

The invention relates to a method for predicting the risk of having CLADin a subject comprising the following steps:

-   -   i) measuring the expression level of POU2AF1 or BLK in a        biological sample obtained from said subject;    -   ii) comparing the expression level of POU2AF1 or BLK with a        predetermined reference value and    -   iii) concluding that the subject is at risk of having CLAD when        the expression level of POU2AF1 or BLK is lower than the        predetermined reference value or concluding that the subject is        not at risk of having CLAD when the expression level of POU2AF1        or BLK is higher than the predetermined reference value. In        particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION

Inventors of the present invention have used a large-scale geneexpression profiling of whole blood to identify early biomarkers ofCLAD. Microarray experiments performed from 80 patients (40 stable and40 BOS) identify 47 genes differentially expressed between the stableand the BOS groups. An independent set of patients (13 stable, 11 BOS)was then used for an external validation by QPCR. POU Class 2Associating Factor 1 (POU2AF1) and B-cell lymphocyte kinase (BLK) geneswere confirmed as predictive markers of BOS more than 6 months beforethe clinical diagnosis.

Method for Predicting the Risk of Having CLAD in a Subject

Accordingly, in a first aspect, the invention relates to a method forpredicting the risk of having CLAD in a subject comprising the followingsteps: i) measuring the expression level of POU2AF1 or BLK in abiological sample obtained from said subject; ii) comparing theexpression level of POU2AF1 or BLK with a predetermined reference valueand iii) concluding that the subject is at risk of having CLAD when theexpression level of POU2AF1 or BLK is lower than the predeterminedreference value or concluding that the subject is not at risk of havingCLAD when the expression level of POU2AF1 or BLK is higher than thepredetermined reference value.

As used herein, the term “predicting” means that the subject to beanalyzed by the method of the invention is allocated either into thegroup of subjects who will have CLAD, or into a group of subjects whowill not have CLAD. Having CLAD referred to in accordance with theinvention, particularly, means that the subject will have higher risk todevelop CLAD. Typically, said risk is elevated as compared to theaverage risk in a cohort of transplanted subjects. In the context of theinvention, the risk of having CLAD in a subject shall be predicted. Theterm “predicting the risk”, as used herein, refers to assessing theprobability according to which the patient as referred to herein willhave CLAD. As will be understood by those skilled in the art, such anassessment is usually not intended to be correct for 100% of thesubjects to be investigated. The term, however, requires that predictioncan be made for a statistically significant portion of subjects in aproper and correct manner. Whether a portion is statisticallysignificant can be determined without further ado by the person skilledin the art using various well known statistic evaluation tools, e.g.,determination of confidence intervals, p-value determination, Student'st-test, Mann-Whitney test etc. Details are found in Dowdy and Wearden,Statistics for Research, John Wiley & Sons, New York 1983. Preferredconfidence intervals are at least 90%, at least 95%, at least 97%, atleast 98% or at least 99%. The p-values are, preferably, 0.1, 0.05,0.01, 0.005, or 0.0001. Preferably, the probability envisaged by theinvention allows that the prediction of an increased risk will becorrect for at least 60%, at least 70%, at least 80%), or at least 90%of the subjects of a given cohort or population. The term, preferably,relates to predicting whether or not there is an increased risk ofhaving CLAD compared to the average risk of CLAD in a population ofsubjects rather than giving a precise probability for the said risk.

As used herein, the term “CLAD” refers to chronic lung allograftdysfunction. CLAD is the main limitation of long term survival afterlung transplantation. The prevalence of CLAD is around 50% at 5 years(35% for the BOS and 15% for the RAS phenotype). Its late diagnosis,based upon the decline of the lung functions, reveals an advanceddegradation of the allograft. Prognosis is poor, with respectively 4 and2 years median survival for BOS and RAS phenotype after disease onset.

In a particular embodiment, the method according to the invention issuitable to predict the risk of having BOS. As used herein, the term“BOS” refers to bronchiolitis obliterans syndrome. It is the main CLADsubtype. It refers to a lung disorder that is mainly associated withchronic allograft dysfunction after lung transplantation. BOS ischaracterized by inflammation and fibrosis of bronchiolar walls thatreduce the diameter of the bronchioles and result in progressive andirreversible airflow obstruction.

In a particular embodiment, the method is suitable to predict the riskof having RAS. As used herein, the term “RAS” refers to restrictiveallograft syndrome (RAS). RAS is characterized by a stair-stepprogression pattern, with tissue damage and fibrotic lesions occurringin the periphery of the lungs (ie, in the visceral pleura, in thealveolar interstitium and in the interlobular septa), resulting in areduction of total lung capacity.

As used herein, the term “subject” refers to any mammals, such as arodent, a feline, a canine, and a primate. Particularly, in the presentinvention, the subject is a human. In a particular embodiment, thesubject is a transplanted subject. As used herein, the term“transplanted subject” also called as grafted subject, refers to asubject who has received an organ transplantation. The term “organtransplantation” refers to the procedure of replacing diseased organs,parts of organs, or tissues by healthy organs or tissues. Thetransplanted organ or tissue can be obtained either from the subjecthimself (=autograft), from another human donor (=allograft) or from ananimal (=xenograft). Transplanted organs may be artificial or natural,whole (such as kidney, heart, lung and liver) or partial (such as heartvalves, lung, skin and bone). In a particular embodiment, the subject isa lung transplanted subject. In particular, said lung transplantedsubject may further have been grafted with the liver or the kidney, ofthe lung donor or of a non-related donor.

As used herein, the term “expression level” refers to the expressionlevel of each of the 2 genes with further other values corresponding tothe clinical parameters. Typically, the expression level of the 2 genesmay be determined by any technology known by a person skilled in theart. In particular, each gene expression level may be measured at thegenomic and/or nucleic and/or protein level. In a particular embodiment,the expression level of gene is determined by measuring the amount ofnucleic acid transcripts of each gene. In another embodiment, theexpression level is determined by measuring the amount of each genecorresponding protein. The amount of nucleic acid transcripts can bemeasured by any technology known by a man skilled in the art. Inparticular, the measure may be carried out directly on an extractedmessenger RNA (mRNA) sample, or on retrotranscribed complementary DNA(cDNA) prepared from extracted mRNA by technologies well-known in theart. From the mRNA or cDNA sample, the amount of nucleic acidtranscripts may be measured using any technology known by a man skilledin the art, including nucleic microarrays, quantitative PCR,microfluidic cards, and hybridization with a labelled probe. In aparticular embodiment, the expression level is determined usingquantitative PCR. Quantitative, or real-time, PCR is a well-known andeasily available technology for those skilled in the art and does notneed a precise description. Methods for determining the quantity of mRNAare well known in the art. For example the nucleic acid contained in thebiological sample is first extracted according to standard methods, forexample using lytic enzymes or chemical solutions or extracted bynucleic-acid-binding resins following the manufacturer's instructions.The extracted mRNA is then detected by hybridization (e. g., Northernblot analysis) and/or amplification (e.g., RT-PCR). Preferablyquantitative or semi-quantitative RT-PCR is preferred. Real-timequantitative or semi-quantitative RT-PCR is particularly advantageous.Other methods of amplification include ligase chain reaction (LCR),transcription-mediated amplification (TMA), strand displacementamplification (SDA) and nucleic acid sequence based amplification(NASBA). Nucleic acids having at least 10 nucleotides and exhibitingsequence complementarity or homology to the mRNA of interest herein findutility as hybridization probes or amplification primers. It isunderstood that such nucleic acids do not need to be identical, but aretypically at least about 80% identical to the homologous region ofcomparable size, more preferably 85% identical and even more preferably90-95% identical. In certain embodiments, it will be advantageous to usenucleic acids in combination with appropriate means, such as adetectable label, for detecting hybridization. A wide variety ofappropriate indicators are known in the art including, fluorescent,radioactive, enzymatic or other ligands (e. g. avidin/biotin). Probestypically comprise single-stranded nucleic acids of between 10 to 1000nucleotides in length, for instance of between 10 and 800, morepreferably of between 15 and 700, typically of between 20 and 500.Primers typically are shorter single-stranded nucleic acids, of between10 to 25 nucleotides in length, designed to perfectly or almostperfectly match a nucleic acid of interest, to be amplified. The probesand primers are “specific” to the nucleic acids they hybridize to, i.e.they preferably hybridize under high stringency hybridization conditions(corresponding to the highest melting temperature Tm, e.g., 50%formamide, 5× or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate). Thenucleic acid primers or probes used in the above amplification anddetection method may be assembled as a kit. Such a kit includesconsensus primers and molecular probes. A kit also includes thecomponents necessary to determine if amplification has occurred. The kitmay also include, for example, PCR buffers and enzymes; positive controlsequences, reaction control primers; and instructions for amplifying anddetecting the specific sequences. In a particular embodiment, the methodof the invention comprises the steps of providing total RNAs extractedfrom a biological sample and subjecting the RNAs to amplification andhybridization to specific probes, more particularly by means of aquantitative or semi-quantitative RT-PCR. In another embodiment, theexpression level is determined by DNA chip analysis. Such DNA chip ornucleic acid microarray consists of different nucleic acid probes thatare chemically attached to a substrate, which can be a microchip, aglass slide or a microsphere-sized bead. A microchip may be constitutedof polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, or nitrocellulose. Probescomprise nucleic acids such as cDNAs or oligonucleotides that may beabout 10 to about 60 base pairs. To determine the expression level, abiological sample from a test subject, optionally first subjected to areverse transcription, is labelled and contacted with the microarray inhybridization conditions, leading to the formation of complexes betweentarget nucleic acids that are complementary to probe sequences attachedto the microarray surface. The labelled hybridized complexes are thendetected and can be quantified or semi-quantified. Labelling may beachieved by various methods, e.g. by using radioactive or fluorescentlabelling. Many variants of the microarray hybridization technology areavailable to the man skilled in the art (see e.g. the review by Hoheisel(16).

As used herein, the term “biological sample” refers to any sampleobtained from a transplanted subject, such as a serum sample, a plasmasample, a urine sample, a blood sample, a lymph sample, or a tissuebiopsy. In a particular embodiment, biological samples for thedetermination of an expression level include samples such as a bloodsample, a lymph sample, or a biopsy. In a particular embodiment, thebiological sample is a blood sample, more particularly, peripheral bloodmononuclear cells (PBMC). Typically, these cells can be extracted fromwhole blood using Ficoll, a hydrophilic polysaccharide that separateslayers of blood, with the PBMC forming a cell ring under a layer ofplasma. Additionally, PBMC can be extracted from whole blood using ahypotonic lysis, which will preferentially lyse red blood cells. Suchprocedures are known to the experts in the art.

As used herein, the term “POU2AF1” refers to POU Class 2 AssociatingFactor 1. The naturally occurring human POU2AF1 gene has a nucleotidesequence as shown in Genbank Accession number NM_006235 and thenaturally occurring human POU2AF1 protein has an aminoacid sequence asshown in Genbank Accession number NP_006226.2. The murine nucleotide andamino acid sequences have also been described (Genbank Accession numbersNM_011136.2 and NP_035266.1).

As used herein, the term “BLK” refers to B-cell lymphocyte kinase. Theprotein BLK encoded by BLK gene and has a role in B-cell receptorsignaling and B-cell development. The naturally occurring human BLK genehas a nucleotide sequence as shown in Genbank Accession numberNM_001715.2 and the naturally occurring human BLK protein has anaminoacid sequence as shown in Genbank Accession number NP_001706.2. Themurine nucleotide and amino acid sequences have also been described(Genbank Accession numbers NM_007549.2 and NP_031575.2).

As used herein, the term “predetermined reference value” refers to athreshold value or a cut-off value. Typically, a “threshold value” or“cut-off value” can be determined experimentally, empirically, ortheoretically. A threshold value can also be arbitrarily selected basedupon the existing experimental and/or clinical conditions, as would berecognized by a person of ordinary skilled in the art. For example,retrospective measurement in properly banked historical subject samplesmay be used in establishing the predetermined reference value. Thethreshold value has to be determined in order to obtain the optimalsensitivity and specificity according to the function of the test andthe benefit/risk balance (clinical consequences of false positive andfalse negative). Typically, the optimal sensitivity and specificity (andso the threshold value) can be determined using a Receiver OperatingCharacteristic (ROC) curve based on experimental data. For example,after determining the expression level of the selected peptide in agroup of reference, one can use algorithmic analysis for the statistictreatment of the expression levels determined in samples to be tested,and thus obtain a classification standard having significance for sampleclassification. The full name of ROC curve is receiver operatorcharacteristic curve, which is also known as receiver operationcharacteristic curve. It is mainly used for clinical biochemicaldiagnostic tests. ROC curve is a comprehensive indicator that reflectsthe continuous variables of true positive rate (sensitivity) and falsepositive rate (1-specificity). It reveals the relationship betweensensitivity and specificity with the image composition method. A seriesof different cut-off values (thresholds or critical values, boundaryvalues between normal and abnormal results of diagnostic test) are setas continuous variables to calculate a series of sensitivity andspecificity values. Then sensitivity is used as the vertical coordinateand specificity is used as the horizontal coordinate to draw a curve.The higher the area under the curve (AUC), the higher is the accuracy ofdiagnosis. On the ROC curve, the point closest to the far upper left ofthe coordinate diagram is a critical point having both high sensitivityand high specificity values. The AUC value of the ROC curve is between1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and betteras AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy islow. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUCis higher than 0.9, the accuracy is high. This algorithmic method ispreferably done with a computer. Existing software or systems in the artmay be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR,MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VI0.0 (DynamicMicrosystems, Inc. Silver Spring, Md., USA), etc.

Method for Preventing the Risk of Having CLAD

In a second aspect, the invention relates to a method for preventing therisk of having CLAD in a subject comprising a step of administering tosaid subject a therapeutically amount of immunosuppressive drugs.

In the context of the invention, the term “preventing the risk” or“prophylactic treatment” as used herein, refers to treatment as well ascurative or disease modifying treatment, including treatment of subjectsat risk of contracting the disease or suspected to have contracted thedisease as well as subjects who are ill or have been diagnosed assuffering from a disease or medical condition, and includes suppressionof clinical relapse. The treatment may be administered to a subjecthaving a medical disorder or who ultimately may acquire the disorder, inorder to prevent, cure, delay the onset of, reduce the severity of, orameliorate one or more symptoms of a disorder or recurring disorder, orin order to prolong the survival of a subject beyond that expected inthe absence of such treatment. By “therapeutic regimen” is meant thepattern of treatment of an illness, e.g., the pattern of dosing usedduring therapy. A therapeutic regimen may include an induction regimenand a maintenance regimen. The phrase “induction regimen” or “inductionperiod” refers to a therapeutic regimen (or the portion of a therapeuticregimen) that is used for the initial treatment of a disease. Thegeneral goal of an induction regimen is to provide a high level of drugto a subject during the initial period of a treatment regimen. Aninduction regimen may employ (in part or in whole) a “loading regimen”,which may include administering a greater dose of the drug than aphysician would employ during a maintenance regimen, administering adrug more frequently than a physician would administer the drug during amaintenance regimen, or both. The phrase “maintenance regimen” or“maintenance period” refers to a therapeutic regimen (or the portion ofa therapeutic regimen) that is used for the maintenance of a subjectduring treatment of an illness, e.g., to keep the subject in remissionfor long periods of time (months or years). A maintenance regimen mayemploy continuous therapy (e.g., administering a drug at a regularintervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy(e.g., interrupted treatment, intermittent treatment, treatment atrelapse, or treatment upon achievement of a particular predeterminedcriteria [e.g., pain, disease manifestation, etc.]).

A “therapeutically effective amount” is intended for a minimal amount ofactive agent which is necessary to impart therapeutic benefit to asubject. For example, a “therapeutically effective amount” to a subjectis such an amount which induces, ameliorates or otherwise causes animprovement in the pathological symptoms, disease progression orphysiological conditions associated with or resistance to succumbing toa disorder.

As used herein, the term “subject” corresponds to the subject asdescribed above. Typically, the subject is a transplanted subject. Moreparticularly, the subject is a lung transplanted subject. In aparticular embodiment, the subject is susceptible to have BOS. Inanother embodiment, the subject is susceptible to have RAS.

As used herein, the term “immunosuppressive drugs” also known asimmunosuppressive agents or antirejection medications are drugs thatinhibit or prevent the activity of immune system. Typically, the subjectis treated with immunosuppressive drugs or other drugs that arecurrently known in the art or that will be identified in the future. Ina particular embodiment, the subject is under immunosuppressivetreatment, which means that the subject is administered with one or moreimmunosuppressive drugs. Immunosuppressive drugs that may be employed intransplantation procedures include corticosteroids, calcineurininhibitors (cyclosporin, tacrolimus), azathioprine, mycophenolatemofetil and tyrosin kinase inhibitors (everolimus, sirolimus). Thesedrugs may be used in monotherapy or in combination therapies. In thecase of lung transplantation, the following immunosuppressive protocolsare usually used. Subjects with primary lung transplantation receive aninduction treatment. Protocols varies largely among centers worldwidebut usually includes either injections of ATG (anti-thymocyte globulin)or basiliximab (other options are anti CD3 and anti CD5 antibodies),high dose of corticosteroids (>1 mg/kg/day), a calcineurin inhibitor anda fourth immunosuppressive treatment (MMF or Azathioprine) or anassociation of high dose of corticosteroids, calcineurin inhibitors anda third immunosuppressive treatment (MMF or azathioprine).Corticotherapy is then progressively tapered to a lifelong lowmaintenance dose (e.g. 5 to 10 mg/day).

In a particular embodiment, the method according to the inventioncomprises i) determining whether the subject is at risk of having CLADby the method as described above and ii) administering to said subject atherapeutically amount of immunosuppressive drugs when the expressionlevel of POU2AF1 or BLK is lower than the predetermined reference value.Typically, the subject is administered with an increase therapeuticallyamount of immunosuppressive drugs.

In a particular embodiment, the method according to the invention issuitable to prevent the risk of having BOS.

A Method for Immunosuppressive Therapy Weaning

In a third aspect, the invention relates to a method for identifying asubject under immunosuppressive therapy as a candidate forimmunosuppressive therapy weaning or minimization, comprising the stepsof: i) determining whether the subject is at risk of having CLAD by themethod as described above; and ii) concluding that the subject iseligible to immunosuppressive therapy weaning or minimization when thesubject is not at risk of CLAD.

In a particular embodiment, the method according to the invention,wherein, the subject is at risk of having BOS.

In a particular embodiment, the method according to the invention,wherein, the subject is at risk of having RAS.

As used herein, the term “immunosuppressive therapy weaning orminimization” refers to the progressive reduction, and optionallyeventually the suppression of an immunosuppressive therapy.

Kit

In another aspect, the present invention relates to a kit fordetermining whether a subject is at risk of having CLAD comprising atleast one reagent for the determination of an expression levelcomprising the following genes: POU2AF1 or BLK.

As used herein, the term “a reagent for the determination of anexpression level” is meant a reagent which specifically allows for thedetermination of said expression level, i.e. a reagent specificallyintended for the specific determination of the expression level of thegenes comprised in the expression profile. This definition excludesgeneric reagents useful for the determination of the expression level ofany gene, such as taq polymerase or an amplification buffer, althoughsuch reagents may also be included in a kit according to the invention.

In some embodiments, the kit according to the invention may compriseinstructions for determining whether a subject is at risk of havingCLAD. The instructions for determining whether a subject is at risk ofhaving CLAD (BOS or RAS) may include at least one reference expressionprofile. In a particular embodiment, at least one reference expressionprofile is a stable expression profile. Alternatively, at least onereference expression profile may be a graft non-tolerant expressionprofile (e.g. expression profile obtained from a healthy subject).

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1A, FIG. 1B and FIG. 1C are diagrams. POU2AF1 as a predictivemarker of Bronchiolitis Obliterans Syndrome. POU2AF1 expressiondetermined by qPCR was compared between stable and BOS patients. (FIG.1A) POU2AF1 expression was lower in BOS patients 6 months or more beforeCLAD diagnosis (stable vs LTP class, p<0.01). (FIG. 1B) ROC curveindicated that POU2AF1 expression discriminated well stable from BOSpatients with an AUC of 0.8322 (95% CI=0.6382 to 1.026). (FIG. 1C)Kaplan-Meier analysis of BOS-free survival categorized by level ofPOU2AF1 expression.

FIG. 2A, FIG. 2B and FIG. 2C are diagrams. BLK as a predictive marker ofBronchiolitis Obliterans Syndrome. (FIG. 2A) BLK expression determinedby QPCR was compared between stable and BOS patients. (FIG. 2B) ROCcurve indicated that BLK expression discriminated well stable from BOSpatients (AUC=0.7797, 95% CI=0.5688 to 0.9907). (FIG. 2C) Kaplan-Meieranalysis of BOS-free survival categorized by level of BLK expression.

EXAMPLE

Material & Methods

Patients

Lung transplant recipients were recruited within the multicentre COLT(Cohort in Lung Transplantation, NCT00980967) study (Comaé de Protectiondes Personnes Ouest 1-Tours, 2009-A00036-51). Study was approved bylocal ethical committee and all participants provided written informedconsent. From September 2009 to October 2013, 1180 patients wereincluded for 1024 transplantation. Inclusion criteria was at least 2years of follow-up unless the diagnosis of CLAD was made before. Theminimum follow up time for stable patients was 3 years. Patients withinsufficient follow up, patients who die or patients retransplantedwithin 2 years were excluded. The eligible patients (n=688) werephenotyped by a blind adjudication committee based upon pulmonaryfunction tests and chest imaging according to ISHLT/ERS/ATS guidelines(1, 19, as already published (20, 21)). Two hundred and sixty-fivepatients were excluded because of other phenotypes or confoundingfactors, 338 stable patients and 85 BOS patients were identified. Stableand BOS patients were then selected based upon sample availability,absence of infection or acute rejection within one month before or afterblood collection and the quality of RNA. For the identification set, 49stables and 40 BOS patients were used. Twenty five independent patients(13 stable and 12 BOS) were included in the validation set.

RNA Isolation

Samples were collected in PAXgene tubes (PreAnalytix, Qiagen), andstored at −80° C. Total RNA was extracted using the PAXgene blood RNAsystem kit with an on-column DNase digestion protocol according to themanufacturer's instructions. Quantity and quality of total RNA weredetermined using a 2100 Bioanalyzer (Agilent TechnologiesIncorporation). Microarray and qPCR analyses were performed on RNA witha RNA integrity number (RIN) above 6.5.

Gene Expression Microarray Analysis

Cyanin-3 and cyanin-5 labelled RNA were prepared with 100 ng of totalRNA using the Two Color Agilent Low Input Quick Amp Labeling Kitfollowing the manufacturer's instructions (Agilent Technologies Inc,Palo Alto, Calif., USA). The labeled cRNA samples were hybridized onSurePrint G3 Human Gene Expression v3 8×60K Microarrays (Agilent). Dataextraction of median feature intensity was performed with FeatureExtraction software v10.7 (Agilent Technologies). In order to removesignal intensity bias between each array, median feature intensitieswere normalized with the lowess (locally weighted scatterplot smoothing)method, then spots for which half of the samples exhibited a signal lessthan the mean of all median signals were removed. A batch effectcorrection was performed with Combat algorithm on the 28,867 remainingspots. Normalized microarray data were deposited in the Gene ExpressionOminbus (GEO) database (accession number GSE94557).

For identification of differential genes, Student's t-test was performedcomparing STA group and each group of interest with bootstrap resampling(1,000 times). Due to bootstrap's pessimistic bias, genes with mean ofp-values inferior to 5% and fold change superior to 1.5 was consideredas differentially expressed. The biological significance of selectedgenes was assessed using GOminer software. Only GO categories enrichedwith a false discovery rate (FDR) inferior to 5% and with at least 5represented genes were selected. The cell type source of differentialgenes was evaluated using the gene set enrichment analysis web toolEnrichr (22).

Quantitative PCR (qPCR) for Microarray Validation

Microarray results were validated by qPCR with a set of independentsamples. After reverse transcription with Superscript III (Invitrogen)real-time quantitative PCR was performed on a Taqman StepOne plus realtime PCR system (Applied Biosystems) using commercially availableprimers: HPRT1 (Hs99999909_m1), β2M(Hs00984230_m1), ACTB(Hs99999903_m1), DEFA4 (Hs01056651_g1), ELANE (Hs00975994_g1), AZU1Hs00156049_m1, FCRL6 (Hs02341772_m1), IGLL5 (Hs04330879_u1), POU2AF1(Hs01573371_m1), CTSL1 (Hs00964650_m1), KRLC3 (Hs01652462_m1), KRLC4(Hs00255338_m1), BLK (Hs01017452_m1), DEFA3 (Hs00414018_m1) and OLFM4(Hs00197437_m1). Samples were run in duplicate and the geometric mean ofquantification cycle values (Cq) for HPRT1, β2M and ACTB was used fornormalization. Relative expression between a sample and a reference wascalculated according to the 2-ΔΔCq method.

Statistics

For QPCR experiments, the non-parametric Whitney test was applied usingGraphPad Prism (Graphpad software, La Jolla, Calif., USA). *p<0.05,**p<0.01, ***p<0.001.

Results

Gene Expression Profiling of Whole Blood from Lung Transplant Recipients

Patients recruited within the multicentre COLT cohort were phenotyped asstable or BOS by a blind adjudication committee based upon pulmonaryfunction tests and chest imaging according to ISHLT/ERS/ATS guidelines(1, 19). Inclusion criteria was at least 2 years of follow-up unless thediagnosis of CLAD was made before. Stable patients display no signs ofCLAD for at least 3 years after lung transplantation. Stable and BOSpatients were selected according to sample availability, absence ofinfection or acute rejection close to blood collection and quality ofRNA. Two independent sets of patients were then prepared for theidentification and the validation experiments. Patients groups werehomogeneous regarding age, sex, BMI, type of transplantation, inductiontreatment and infection and rejection events.

Since patients were longitudinally followed, several blood samples atdifferent time-point post transplantation could be obtained from onerecipient. We thus organised samples to defined three classes dependingon the time between blood collection and CLAD diagnosis (defined as thetime-point with a decline of ≥20% in FEV1 from the baseline). Long-termoutcome prediction (LTP) class combined blood samples collected morethan 12 months before CLAD diagnosis; medium-term outcome prediction(MTP) class combined blood samples collected within the 12 months thatprecede CLAD diagnosis and samples collected at or after CLAD diagnosiswere incorporated in the diagnosis class (D). No patient duplicates wereincluded within each of these three classes. Total RNA extracted fromblood samples collected or 12 months after lung transplantation wereused for stable patients.

Identification of Gene Signatures Associated with CLAD

For gene expression microarray, RNA from 40 stable patients werecompared with 65 RNA samples from 40 BOS patients (LTP class, n=18; MTPclass, n=21 and D class, n=26). Gene expression profiling identified atotal of 47 genes differently expressed between stable and BOS groups.Comparing the stable group with the D group, we highlighted 20 uniquegenes (26 probes) with significant differential expression and foldchange superior to 1.5, allowing a partial discrimination of stable withD in principal component analysis. GO analysis revealed 6 genesassociated with biological defense response (e.g. GO:0009617, responseto bacterium, FDR<0.0001, GO:0006952, defense response, FDR=0.0018),namely alkaline phosphatase, liver/bone/kidney (ALPL, FC=0.40),azurocidin 1 (AZU1, FC=0.52), defensin alpha 3 (DEFA3, FC=0.41),defensin alpha 4 (DEFA4, FC=0.44), elastase, neutrophil expressed (ELANEFC=0.50) and peptidoglycan recognition protein 1 (PGLYRP1, FC=0.52).

Comparison between the stable and the MTP groups pinpointed 13 genes (16probes) differentially expressed. Principal component analysis show amoderate discrimination between stable and MTP groups. While GO analysisshowed no enrichment of biological ontology, likely due to the lownumber of genes, cell origin analysis using Enrichir enrichment analysishighlighted 3 genes significantly associated with B cells, i.e. Blymphoid tyrosine kinase (BLK, FC=0.62), chemokine (C-X-C motif)receptor 5 (CXCR5, FC=0.64) and POU class 2 associating factor 1(POU2AF1, FC=0.56). Interestingly, unsupervised hierarchical clusteringrevealed that these 3 genes resided in the same cluster, along withB-cell related genes such as CD19, MS4A1, BANK1 and C40, suggesting thepotential association of B-cell related gene expression with MTP.

Finally, 24 genes (48 probes) were identified when comparing stable withLTP groups. Stable and LTP groups were discriminated in principalcomponent analysis (. GO analysis highlighted the enrichment of 7 genesrelated to the immune system (GO:0006955, immune response, FDR=0.022;GO:0002376 immune system process, FDR=0.039 and GO:0006952 defenseresponse, FDR=0.073). Enrichr analysis stressed the enrichment of genesrelated to CD56+NK cells including granzyme A (GZMA, FC=1.62), granzymeB (GZMB, FC=1.67) and the killer cell lectin-like receptor subfamily C,member 3 (KLRC3, FC=1.50) and 4 (KLRC4, FC=1.56). Interestingly, BLK,immunoglobulin lambda-like polypeptide 5 (IGLL5) and POU2AF1 wereassociated with both MTP and LTP suggesting their potential aspredictive biomarkers.

Validation of POU2AF1 and BLK as Predictive Biomarkers of CLAD

Based on their predictive values, biological functions, p-value and FCsuperior to 1.5, we selected 8 genes that were then tested by qPCR on anindependent set of patients. We investigated here the overall predictivevalue of the selected genes, irrespective of time between bloodcollection and CLAD diagnosis, by pooling LTP and MTP class samples.qPCR experiments were performed on 13 stables samples and 11 BOSsamples. Downregulation of POU2AF1 expression 6 months or more beforeCLAD diagnosis was validated by QPCR (p-value<0.01, FC=0.51) (FIG. 1A).ROC curve indicated that POU2AF1 expression discriminated well stablefrom BOS patients with an AUC of 0.8322 (95% CI=0.6382 to 1.026) (FIG.1B). Noteworthy, expression of POU2AF1 in stable patients was constantbetween V3 and V4 (6 and 12 months post transplantation). Regarding BLKexpression, we noted a reduced level of BLK expression in the BOS group(p<0.05, FC=0.56), 6 months or more before CLAD was diagnosed (FIG. 2A).ROC curve show a fair discrimination between the two groups of patients(AUC=0.7797, 95% CI=0.5688 to 0.9907) (FIG. 2B). As for POU2AF1,expression of BLK in stable patient was constant in time. Thedifferential expression of the other 6 selected genes were not confirmedby qPCR. We finally performed Kaplan-Meier analyses to investigate theBOS free survival regarding POU2AF1 (FIG. 1C) and BLK (FIG. 2C)expression. The levels of POU2AF1 or BLK under 0.45 or 0.505respectively reduced significantly the likelihood of BOS-free survivalafter lung transplantation.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

-   1. Meyer K C, Raghu G, Verleden G M, Corris P A, Aurora P, Wilson K    C, Brozek J, Glanville A R, ISHLT/ATS/ERS BOS Task Force Committee,    ISHLT/ATS/ERS BOS Task Force Committee. An international    ISHLT/ATS/ERS clinical practice guideline: diagnosis and management    of bronchiolitis obliterans syndrome. Eur Respir J 2014;    44:1479-1503.-   2. Koutsokera A, Royer P-J, Fritz A, Benden C, Tissot A, Aubert J-D,    Antonietti J-P, Botturi-Cavaillés K, Magnan A, Pison C, Nicod L P.    Risk factors for chronic lung allograft dysfunction (CLAD) in the    SysCLAD cohort. Eur Respir J 2015; 46:PA1800.-   3. Sato M. Chronic lung allograft dysfunction after lung    transplantation: the moving target. Gen Thorac Cardiovasc Surg 2013;    61:67-78.-   4. Sato M, Waddell T K, Wagnetz U, Roberts H C, Hwang D M, Haroon A,    Wagnetz D, Chaparro C, Singer L G, Hutcheon M A, Keshavjee S.    Restrictive allograft syndrome (RAS): a novel form of chronic lung    allograft dysfunction. J Heart Lung Transplant 2011; 30:735-742.-   5. Sato M, Keshavjee S. Bronchiolitis obliterans syndrome:    alloimmune-dependent and -independent injury with aberrant tissue    remodeling. Semin Thorac Cardiovasc Surg 2008; 20:173-182.-   6. Devouassoux G, Drouet C, Pin I, Brambilla C, Brambilla E, Colle    P-E, Pison C, Grenoble Lung Transplant Group. Alveolar neutrophilia    is a predictor for the bronchiolitis obliterans syndrome, and    increases with degree of severity. Transpl Immunol 2002; 10:303-310.-   7. Neurohr C, Huppmann P, Samweber B, Leuschner S, Zimmermann G,    Leuchte H, Baumgartner R, Hatz R, Frey L, Ueberfuhr P, Bittmann I,    Behr J, Munich Lung Transplant Group. Prognostic value of    bronchoalveolar lavage neutrophilia in stable lung transplant    recipients. J Heart Lung Transplant Off Publ Int Soc Heart    Transplant 2009; 28:468-474.-   8. Hubner R H, Meffert S, Mundt U, Böttcher H, Freitag S, El    Mokhtari N E, Pufe T, Hirt S, Folsch U R, Bewig B. Matrix    metalloproteinase-9 in bronchiolitis obliterans syndrome after lung    transplantation. Eur Respir J 2005; 25:494-501.-   9. Bhorade S M, Chen H, Molinero L, Liao C, Garrity E R, Vigneswaran    W T, Shilling R, Sperling A, Chong A, Alegre M-L. Decreased    percentage of CD4+FoxP3+ cells in bronchoalveolar lavage from lung    transplant recipients correlates with development of bronchiolitis    obliterans syndrome. Transplantation 2010; 90:540-546.-   10. Reynaud-Gaubert M, Marin V, Thirion X, Farnarier C, Thomas P,    Badier M, Bongrand P, Giudicelli R, Fuentes P. Upregulation of    chemokines in bronchoalveolar lavage fluid as a predictive marker of    post-transplant airway obliteration. J Heart Lung Transplant 2002;    21:721-730.-   11. Jonigk D, Izykowski N, Rische J, Braubach P, Kühnel M, Warnecke    G, Lippmann T, Kreipe H, Haverich A, Welte T, Gottlieb J, Laenger F.    Molecular Profiling in Lung Biopsies of Human Pulmonary Allografts    to Predict Chronic Lung Allograft Dysfunction. Am J Pathol 2015;    185:3178-3188.-   12. Shah R J, Bellamy S L, Lee J C, Cantu E, Diamond J M,    Mangalmurti N, Kawut S M, Ware L B, Christie J D. Early plasma    soluble receptor for advanced glycation end-product levels are    associated with bronchiolitis obliterans syndrome. Am J Transplant    Off J Am Soc Transplant Am Soc Transpl Surg 2013; 13:754-759.-   13. Salama M, Jaksch P, Andrukhova O, Taghavi S, Klepetko W,    Aharinejad S. Endothelin-1 is a useful biomarker for early detection    of bronchiolitis obliterans in lung transplant recipients. J Thorac    Cardiovasc Surg 2010; 140:1422-1427.-   14. Paantjens A W M, Kwakkel-van Erp J M, Van Ginkel W G J, Van    Kessel D A, Van Den Bosch J M M, Van De Graaf E A, Often H G. Serum    thymus and activation regulated chemokine levels post-lung    transplantation as a predictor for the bronchiolitis obliterans    syndrome. Clin Exp Immunol 2008; 154:202-208.-   15. LaPar D J, Burdick M D, Emaminia A, Harris D A, Stricter B A,    Liu L, Robbins M, Kron I L, Stricter R M, Lau C L. Circulating    fibrocytes correlate with bronchiolitis obliterans syndrome    development after lung transplantation: a novel clinical biomarker.    Ann Thorac Surg 2011; 92:470-477.-   16. Hoheisel J D. Microarray technology: beyond transcript profiling    and genotype analysis. Nat Rev Genet 2006; 7:200-210.-   17. Chesné J, Danger R, Botturi K, Reynaud-Gaubert M, Mussot S,    Stern M, Danner-Boucher I, Mornex J-F, Pison C, Dromer C, Kessler R,    Dahan M, Brugiére O, Le Pavec J, Perros F, Humbert M, Gomez C,    Brouard S, Magnan A, COLT Consortium.

Systematic analysis of blood cell transcriptome in end-stage chronicrespiratory diseases. PloS One 2014; 9:e109291.

-   18. Herazo-Maya J D, Noth I, Duncan S R, Kim S, Ma S-F, Tseng G C,    Feingold E, Juan-Guardela B M, Richards T J, Lussier Y, Huang Y, Vij    R, Lindell K O, Xue J, Gibson K F, Shapiro S D, Garcia J G N,    Kaminski N. Peripheral blood mononuclear cell gene expression    profiles predict poor outcome in idiopathic pulmonary fibrosis. Sci    Transl Med 2013; 5:205ra136.-   19. Verleden G M, Raghu G, Meyer K C, Glanville A R, Corris P. A new    classification system for chronic lung allograft dysfunction. J    Heart Lung Transplant 2014; 33:127-133.-   20. Koutsokera A, Royer P J, Antonietti J P, Fritz A, Benden C,    Aubert J D, Tissot A, Botturi K, Roux A, Reynaud-Gaubert M L,    Kessler R, Dromer C, Mussot S, Mal H, Mornex J-F, Guillemain R,    Knoop C, Dahan M, Soccal P M, Claustre J, Sage E, Gomez C, Magnan A,    Pison C, Nicod L P, Consortium T S. Development of a Multivariate    Prediction Model for Early-Onset Bronchiolitis Obliterans Syndrome    and Restrictive Allograft Syndrome in Lung Transplantation. Front    Med 2017; 4.-   21. Pain M, Royer P-J, Loy J, Girardeau A, Tissot A, Lacoste P, Roux    A, Reynaud-Gaubert M, Kessler R, Mussot S, Dromer C, Brugiére O,    Mornex J-F, Guillemain R, Dahan M, Knoop C, Botturi K, Pison C,    Danger R, Brouard S, Magnan A, COLT Consortium. T Cells Promote    Bronchial Epithelial Cell Secretion of Matrix Metalloproteinase-9    via a C—C Chemokine Receptor Type 2 Pathway: Implications for    Chronic Lung Allograft Dysfunction. Am J Transplant Off J Am Soc    Transplant Am Soc Transpl Surg 2016; doi:10.1111/ajt.14166.-   22. Kuleshov M V, Jones M R, Rouillard A D, Fernandez N F, Duan Q,    Wang Z, Koplev S, Jenkins S L, Jagodnik K M, Lachmann A, McDermott M    G, Monteiro C D, Gundersen G W, Ma'ayan A. Enrichr: a comprehensive    gene set enrichment analysis web server 2016 update. Nucleic Acids    Res 2016; 44:W90-97.

1. A prognostic method for determining whether a subject is at risk ofhaving Chronic Lung Allograft Dysfunction (CLAD) and preventing therisk, comprising: measuring the expression level of POU2AF1 in abiological sample obtained from the subject; comparing the expressionlevel of POU2AF1 with a predetermined reference value; concluding thatthe subject is at risk of having CLAD when the expression level ofPOU2AF1 is lower than the predetermined reference value; andadministering to the subject determined to be at risk a therapeuticamount of immunosuppressive drugs to prevent CLAD.
 2. The methodaccording to claim 1, wherein the subject is a subject who has had alung transplant.
 3. The method according to claim 1 further comprising:determining whether the subject is at risk of having BronchiolitisObliterans Syndrome (BOS) when the expression level of POU2AF1 is lowerthan the predetermined reference value.
 4. The method according to claim1 further comprising: determining whether the subject is at risk ofhaving Restrictive Allograft Syndrome (RAS) when the expression level ofPOU2AF1 is lower than the predetermined reference value.
 5. A method forimmunosuppressive therapy weaning or minimization for a subject underimmunosuppressive therapy, comprising: measuring the expression level ofPOU2AF1 in a biological sample obtained from the subject; comparing theexpression level of POU2AF1 with a predetermined reference value;concluding that the subject is not at risk of having CLAD when theexpression level of POU2AF1 is higher than the predetermined referencevalue; concluding that the subject is eligible for immunosuppressivetherapy weaning or minimization; and then administering to the subjectdetermined not to be at risk of having CLAD a progressive reduction of atherapeutic amount of immunosuppressive drugs to prevent CLAD.
 6. Themethod according to claim 1, wherein, the subject is susceptible to haveBOS.
 7. The method according to claim 1, wherein, the subject issusceptible to have RAS.